Secretion of a cytoplasmic lectin from Xenopus laevis skin

The skin of Xenopus laevis contains a soluble beta-galactoside-binding lectin with a approximately 16,000-mol-wt subunit. It resembles similar lectins purified from a variety of tissues from other vertebrates, and differs from two other soluble X. laevis lectins from oocytes and serum that bind alpha-galactosides. The skin lectin is concentrated in the cytoplasm of granular gland and mucous gland cells, as demonstrated by immunohistochemistry with the electron microscope. Upon injection with epinephrine, there is massive secretion of the cytoplasmic lectin from the granular gland cells.     

Peptide C-terminal alpha-amidating enzyme purified to homogeneity from Xenopus laevis skin

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. In this study, we have developed a simple and sensitive assay for monitoring alpha-amidating activity by using radioiodinated Ac-Tyr-Phe-Gly as a substrate. By utilizing this assay, an alpha-amidating enzyme was first purified to homogeneity from Xenopus laevis skin. The purified enzyme has a single polypeptide chain with an apparent molecular weight of 39,000 and its N-terminal sequence was determined as Ser-Leu-Ser-. The enzyme converts several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides. The enzyme activity, with an optimal pH 6-7, was dependent on the copper ion and ascorbate. In the presence of 0.25 mM ascorbate, the enzyme exhibited a Km of 0.35 microM and a Vmax of 1.9 nmol/microgram/h for Ac-Tyr-Phe-Gly.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Sequence and specificity of a soluble lactose-binding lectin from Xenopus laevis skin.

A 16-kDa lactose-binding lectin comprises 5% or more of the soluble protein in Xenopus laevis skin. This lectin is mainly localized in the cytoplasm of granular gland cells. In response to stress, the lectin along with a variety of toxic and antibiotic peptides are released onto the skin surface by holocrine secretion. We have purified the lectin, sequenced tryptic peptides using tandem mass spectrometry and Edman degradation, and isolated full-length cDNA using a deduced oligonucleotide. Comparison of the cDNA and peptide sequences revealed expression of at least two isolectins, which differ in sequence at only two or three amino acids. Comparison of cDNA with complementary message by ribonuclease protection confirmed expression in approximately equal abundance of two nearly identical messages. The major soluble lactose-binding lectin expressed in Xenopus muscle is composed of these same isolectins, but at 100-fold lower levels. Similarities and distinctions in sequence and carbohydrate-binding specificity indicate that this lectin is a novel member of a family of soluble lactose-binding lectins expressed in a wide range of vertebrate tissues.     

Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions

Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human β[1-40]-amyloid peptide and related peptides. Cleavage of the wild type β[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His¹⁴-Glu¹⁵ bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of β amyloid peptide of Xenopus laevis is discussed.     

Characterization of a proteolytic enzyme in the skin secretions of Xenopus laevis

Enzymes present in the skin secretions of Xenopus laevis were fractionated by ion exchange chromatography. One of the proteases obtained was found to catalyse cleavage on the COOH-side of peptide sequences containing consecutive hydrophobic and basic residues. Evidence is presented that the enzyme is a cysteine protease with an optimum pH of 5.0 to 6.0. The characteristic specificity of this enzyme suggests that it may fulfil a role in propeptide processing.     

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.     

An unusual repetitive structure of caerulein mRNA from the skin of Xenopus laevis

The nucleotide sequence of a 784-bp segment of cloned caerulein mRNA obtained from the skin of Xenopus laevis was determined. It codes for five heterogeneous procaerulein peptides interspersed with three 147-bp intercaerulein segments (ICS). The ICSs contain six inverted repeats and five eukaryotic enhancer-like sequences. Evidence for the presence of multiple forms of caerulein mRNA is presented.     

A new repetitive protein from Xenopus laevis skin highly homologous to pancreatic spasmolytic polypeptide

A cDNA sequence has been used to derive the precursor structure of a highly repetitive protein in Xenopus laevis skin. From the sequence of a whole family of secretory proteins can be predicted containing a classical hydrophobic signal sequence at the NH2-terminal end of the precursor. The proteins contain four domains with high homology to porcine pancreatic spasmolytic polypeptide. These four cysteine-rich, presumably physiologically active domains are separated in the molecule by a repetitive element, locating two such domains to the NH2 terminus of the precursor protein and the remaining two to the COOH-terminal end. The separating spacer consists of very unusual, precise, threonine and proline-rich repeats containing 9 residues which could be targets for extensive O-glycosylation. Additionally, processing at two pairs of basic residues is suggested to liberate two polypeptides ("spasmolysins") and "spasmolysin-glycoprotein."     

A melanocyte-stimulating substance in the skin secretion of Xenopus laevis

Summary The melanocyte-stimulating substance, found in the dorsal skin secretion of Xenopus laevis, has been identified as 5-hydroxytryptamine (serotonin).     

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate

Using a monoclonal antibody against the entire C-terminal end of human APP₆₉₅ (643–695 sequence) and a monoclonal antibody directed against human β[1–40] amyloid peptide (βA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of βA. According to the nature of N- and C-terminal amino acids we identified endogenous β-, γ-, ε-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the β-amyloid sequence at same sites as those observed in human βA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study βA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human βA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.     

Isolation and characterization of three novel serine protease genes from Xenopus laevis

Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis. These Xenopus cDNAs encode trypsin-like serine proteases and are designated Xenopus embryonic serine protease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.     

Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis

The skin secretion of the frog Xenopus laevis has been fractionated by reverse-phase HPLC and the most polar components studied by fast-atom-bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

Spermiogenesis in Xenopus laevis: from late spermatids to spermatozoa

Spermatogenesis is a complex morphogenetic process in which microfilaments and microtubules have been shown to play an important role. The last steps of Xenopus spermatogenesis, i.e., the corkscrew shaping of the sperm head, have been followed to study actin and microtubule distribution by conventional and immunoelectron microscopy. During sperm head morphogenesis, actin is absent in the elongating spermatids, but it is present in the Sertoli cells where results localized at the periphery of their cytoplasm that surrounds the developing germ cells. Sertoli cell actin and microtubules may assist the elongation and the shaping of the spermatids and function in maintaining the Sertoli-spermatid association.     

Isolation of a dipeptidyl aminopeptidase, a putative processing enzyme, from skin secretion of Xenopus laevis

A dipeptidyl aminopeptidase has been purified to apparent homogeneity from skin secretion of Xenopus laevis. This enzyme is a glycoprotein with a molecular mass of about 98 kDa. It hydrolyzes a variety of dipeptidyl-p-nitroanilides and oligopeptides containing proline, alanine or glycine as the second amino acid and is inhibited by diisopropylfluorophosphate. The pH optimum was found to be around 8, while at pH 6, substrates were cleaved at about one-third of the maximal rate. This dipeptidyl aminopeptidase has the specificity required for the cleavage of amino-terminal extensions preceding the sequence of caerulein and xenopsin in their respective precursors.     

A ventrally localized inhibitor of melanization in Xenopus laevis skin

Melanophores normally differentiate in dorsal but not in ventral skin of Xenopus laevis. We have sought factors which might regulate this differentiation pattern, and we have obtained a putative melanization inhibiting factor (MIF) from ventral but not from dorsal skin. Preliminary studies reveal that MIF is destroyed by heat or trypsin treatment, indicating its protein composition, and has a molecular weight in the range of 300 kDa. The effects of MIF on the differentiation of neural crest derivatives to melanophores were examined in vitro in the presence of tyrosine and fetal calf serum (FCS). Tyrosine enhances melanophore differentiation in vitro at concentrations equivalent to those estimated in adult Xenopus blood plasma (20 microM). FCS also stimulates melanization, by way of materials other than the tyrosine contained in FCS. MIF strongly inhibits outgrowth and melanization of neural crest cells from neural tube explants. MIF also inhibits the differentiation of melanoblasts contained in cultured explants of ventral skin. Inhibition of melanization or melanophore differentiation by MIF occurs even in the presence of L-tyrosine and/or FCS. We suggest that MIF plays an important role in the establishment of dorso-ventral pigment patterns in amphibia.